In both prokaryotic and eukaryotic cells, some proteins are synthesized as longer precursors. These precursors require one or more proteolytic cleavages to generate an active, mature molecule. The precursor forms contain additional amino acids known as pre-and pro-sequences. Pre- and pro-sequences can be found singly or in combination in precursor molecules.
The pre-sequence is located at the N-terminus of the polypeptide chain and has been determined to be necessary for secretion and membrane localization. Generally, the pre-sequence (signal peptide) is 20 to 30 amino acids in length and contains a high content of hydrophobic residues. This precursor exists transiently. When the growing peptide chain is long enough for the signal peptide to extend beyond the ribosome, a cellular signal recognition particle binds the signal and the resultant particle/ribosome complex moves to the cell membrane. During binding of the complex to the membrane receptor, the signal recognition particle is displaced. As translation continues, the pre-sequence passes through the membrane and is followed by the rest of the nascent polypeptide chain. At a point when the protein is well inserted into the membrane, the signal sequence is cleaved off. After translation is complete, the protein has either passed entirely through the membrane (secretion) or is localized there (membrane bound). Removal of the signal sequence is the only cleavage necessary to generate the mature forms of secretory proteins like placental lactogen, lysozyme, ovomucoid, growth hormone and the viral membrane protein, VSV glycoprotein.
Most proteins, however, contain the additional pro-sequence. After cleavage of the pre-sequence, the resultant proprotein or prohormone exists as a stable precursor. Cleavage to generate the mature, active molecule may not occur until it is packaged in secretory vesicles. Many cells secrete toxins or potentially hazardous enzymes. It is thought that this delay in the production of an active protein protects the producing cell from the possible deleterious effects of the polypeptide produced. Examples of proteins which initially exist in the pro-form are albumin, insulin, parathyroid hormone and influenza virus hemagglutinin.
The function of the pro-sequence has not been well established. It is thought that this sequence may be required for the association of the pro-enzyme with the cell before release of the active enzyme into the medium and/or for guiding the proper folding of the protein into its active conformation. Recently, in the case of subtilisin E from B. subtilis, the covalently attached pro-sequence has been determined to be essential in guiding the proper folding of the protein to give active enzyme. (Ikemura et al, 1987, Ikemura and Inouye, 1988).
Many proteins have been cloned and overexpressed in heterologous systems. In some instances, these cloned proteins can be purified in a biologically active form using slight modifications of purification protocols worked out for natural sources of the protein. In other cases, the proteins produced exhibit diminished biological activity. Microscopic analysis of the host cell has indicated that large aggregates of these inactive proteins, called inclusion bodies, may form. The polypeptides exist in a non-native, inactive state within these aggregates. The aggregates are easily sedimented and can be dissociated by denaturing agents such as urea or guanidine hydrochloride. After dissociation, the protein must be renatured into its correct conformation. Successful renaturation techniques vary and are largely determined empirically. The problems associated with proper protein folding and the need for a more systematic approach has been recognized by the biotechnology industry and has spurred considerable research on protein folding (King, 1989).
It has been observed that in order to achieve maximum biological activity some proteins require their leader sequences to be cloned as well. It is thought that their inactivity which results from lack of a leader sequence is due to improper folding. The remedy of providing the leader sequence results in a final product having more amino acids than the natural product, where, in E. coli for example, the host cell lacks the ability to process these sequences off of the precursor molecule. The resultant protein may remain inactive until the prosequence is removed. Methods which exist to effect this removal have proved to be cumbersome.
The present invention provides a method for activation of polypeptides expressed without their pro-sequence or which have been partially or totally denatured in which an exogenous peptide sequence is added in trans to said polypeptides. The invention also provides the biologically active peptides.